The processes of stem cell self-renewal and differentiation are regulated in large part by specialized niches, structurally complex microenvironments that present their resident stem cells with numerous cues in the form of soluble factors, extracellular matrix (ECM), and juxtacrine factors from neighboring cells. Within the adult mammalian brain, for example, neural stem cells (NSCs) in the hippocampus continuously divide to give rise to new neurons that play roles in learning and memory, and these cells are regulated by growth factors, morphogens, ECM, and juxtacrine signals from neighboring astrocytes. Gaining deeper insights into the mechanisms through which these niches regulate stem cells can aid in the development of in vitro biomaterials culture systems for expanding and differentiating stem cells in translational medicine applications. It is now widely appreciated that in addition to its biochemical properties, the mechanical properties of the niche (e.g., stiffness) can also powerfully regulate stem cell behavior, and indeed we recently showed that this true of NSCs. However, in general the field lacks key molecular and mechanistic information about how stem cells process such mechanical cues at the cell-ECM interface to give rise to changes in cell fate, how these mechanotransductive signals interface with transcriptional events traditionally understood to control processes such as neurogenesis, and whether mechanotransductive signaling can also control neurogenesis in vivo. In this proposal we will address all of these open questions. Aim 1 will investigate the dynamics of mechanotransduction to cellular adhesion receptors and the cytoskeleton. Specifically, we will conduct biophysical and biochemical measurements to analyze how mechanical information from the cellular microenvironment is propagated through cells - including adhesion receptors, focal adhesion proteins, and nonmuscle myosin II - as cell fate decisions are made. In addition, Aim 2 will investigate how substrate stiffness impacts the activation of NeuroD to control neuronal differentiation. We will do this by quantifying the dynamics by which the gene encoding a key proneural transcription factor receives and integrates upstream mechanical signals as cells commit to a neuronal fate. An innovative tool in this aim, new to this revised application, i the use of synthetic ECMs whose stiffness may be dynamically and reversibly switched. This revised application also includes new data demonstrating our ability to control neurogenesis in vivo by genetically manipulating mechanotransductive signals in a rat model. Thus, in both aims we will apply this capability to determine whether signaling effectors implicated in mechanosensitive fate choice in vitro also regulate this process in vivo. In summary, this proposal blends stem cell biology, mechanobiology, and materials synthesis to develop quantitative, mechanistic insights into stem cell mechanoregulation, with implications for both basic stem cell biology and the future development of advanced biomaterials systems for regenerative medicine.